Control mechanisms governing protein-protein interactions in assemblies

Control mechanismsgoverning proteinprotein interactions in assemblies. E. Kellenberger The study of the assembly and maturation of bacteriophages allows us to investigate biological control mechanisms which act on the level of proteins. Such studies complement those on the now well known mechanisms which operate on the level of switching on or switching off the expression of genes. The present paper explains what has been learned about how protein interactions are stimulated sequentually by conformational changes. It also discusses our understanding of how the shapeof a virus is determined by the proteins involved.

Introduction

It is well established that the control and regulation of cellular functions occur on the following two levels: (a) The presence,amount, or absenceof enzymes or other proteins which are the ultimate geneproducts is controlled by turning on or off the transcription, or possibly the translation, during protein biosynthesis (figure 1). Mechanisms at this level are usedin cell differentiation and in the regulation of metabolic pathways. (b) The functions of enzymes and other proteins may become activated by induced conformational changes. Such a mechanism is involved, for example, in oxygen uptake and release by haemoglobin and in the action of many enzymes. This mechanism also explains how the functions of the manifold proteins contained in biological membranesare controlled. These govern, for example, the active transport of metabolites from a low external concentration to a much higher internal one; they control the flow of ions and small moleculesthrough gates;and also pump ions selectively to the interior. Ion pumps are thought to be involved in signal transmission in nerves and also in the function of rnitochondria, chloroplasts, and the retina. The multiplication of viruses in a host cell is controlled on both levels. Bacteriophage A has provided the textbook example for a well studied set of transcriptional controls, so that we need not to consider it here. In what follows, we would like to emphasise the control mechanisms which operate at the level of the gene products and are the only ones governing the assembly and maturation of virus particles. The study of phage morphogenesishas provided unique insights into these control mechanismsand there is still an enormous potential for further detailed investigations. After describing some characteristic features of virus multiplication and morphogenesiswe will chooseexamples illustrating the knowledge that has been acquired on some of the mechanismsgoverning the sequenceof events which take place in assemblingand maturing a virus particle. We will show that the proteins composing a particle undergo profound conformational changes, which determine the exact moment at which the next event occurs. Examples are the partial in situ proteolytic cleavage of constituent proteins or the binding of an additional protein species taken out of the pool of soluble protein subunits. We will see that individual protein molecules destined to interact very Edward

Kellenberger.

Ph.D.

His Ph.D. ~n physics was gamed with a thesis about structural studies on the bacterial nucleus. and then concentrated on problems related to the reproduction of bacterial virus. As a corollary to these studies he was involved in many methodological developments in btological electron microscopy. Edward Kellenberger is presently Professor of Microbiology at the University of Easel, Biozentrum. and formerly Professor of Biophysics at the University of Geneva in Switzerland.

2

strongly in the assembledmature particle do not do so at all when still in the form of soluble subunits. In this state each gene product ‘patiently’ awaits involvement until a precursor particle has becomeready for interaction. We shall then discuss the available experimental results relative to the determination of the form of viruses. We will seethat here also protein-protein interactions are essential. Frequently a structural part of a precursor particle acts transiently as a sort of internal scaffold required to achieve the correct form of the virus shell. But we shall learn also that some essential parts of the molecular mechanismsof form determination are still not yet elucidated, although the experimental possibilities for doing so are excellent. The multiplication

of a virus

For the reader not familiar with current ideas of the nature viruses, I will summarise the events involved in their reproduction. Viruses are able to infect only specific types of cells, called hosts. These possessspecific receptors on their surface, which allow attachment of the virus and its complete or partial penetration into the cell. Many different mechanismsfor this invasion are known: they all have in common that the viral nucleic acid which enters the cell is then able to use the existing cellular apparatus for protein synthesis. The genetic message of the virus leads to formation of specific enzymes which allow preferential replication of the viral nucleic acid. Later, other viral genes becomeexpressedwhich produce the proteins necessaryfor building new virus particles. Viral nucleic acid is withdrawn from the existing pool, and together with the viral proteins particles known as virions are built. As we will seebelow, many steps are involved in this morphogenesisof virions. During all these events, the cell stays intact, and its metabolic systemcontinues to function. The mechanismsby which the virus progeny is released from the cell differ according to the species.In a few cases, the virion can go out of the infected cell, without harming it: virions are in some way secreted continuously. In most cases,however, the cells-which are full of virions-lyse; that is, they burst and so releasethe virions, which are then ready to infect new cells. All cycles of virus multiplication follow this general schemeof invasion, replication of nucleic acid, assemblyof virions, and release, summarised in figure 2. For completeness, and because of its biological interest, mention must be made of an additional property of many viruses, infection with which does not necessarily lead to multiplication. With numerous virus speciesthe interaction with the host leadsto the integration of the viral nucleic acid into its genetic apparatus. The cell, now called virogen (lysogen with bacteria), starts to divide normally again. The integrated piece of viral information confers some new

properties on the cell. For example lysogenic bacteria might be induced to produce infective virions when treated with chemicalsor irradiated with ultraviolet light or X-rays.

DNA u

b

+

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DNA

translation

Ribosome /

Protein biosynthesis

transcription Figure 1 Summary of DNA replication and of the mechanisms involved in protein biosynthesis. The upper part of the figure visualises the process of ‘semiconservative’ replication of DNA (Desoxy-ribonucleic acid), by which the genetic information is preserved and copied. The horizontal bars represent a pair of purine and pyrimidine bases: the dark line symbolises the ‘backbone’ of desoxyribose which is linked together by phosphate radicals. The lower part shows very schematically the process of protein biosynthesis. Single stranded mRNA (messenger ribonucleic acid) is synthesised on DNA as template and copies the information, which is carried by one of the strands of the double-stranded DNA. The sequence of bases determine the sequence of amino-acids in the polypeptide by means of the genetic code. The mRNA leaves the nucleus and goes to the rlbosomes in the cytoplasm; it attaches to them and, by the mediation of another RNA species (tRNA), the correct amino-acids are associated with the code word in the mRNA. The ribosomes move along the mRNA and thus one amino acid after the other becomes ordered into the linear peptide chain. Enzymes promote the covalent linkage between the amino acids. The linear peptide chain (primary structure) then folds to form the secondary and tertiary structure of the protein.

The multiplication cycles of different viruses show many small differences. In most cases the cell continues to produce cellular proteins besidesthose coded by the virus. Only in a few casesdoesthe virus stop the expressionof the information contained in the cellular nucleus; sometimesit may then even destroy the latter and useits building blocks for its own synthesis. This occurs, for example, with bacteriophage T4 (figure 3) which we will usefrequently as an example in what follows. It is obvious that in

experimental work such a virus has the advantage that all proteins synthesisedafter infection are of viral origin. From what has beensaid above, it is clear that the events in a cell infected with a DNA-virus can conveniently be subdivided into two main classes,namely early and late functions. The early functions concern host-virus interactions and, in the event that a multiplicative cycle is chosen,they govern the replication of the viral nucleic acid. The early functions are controlled at the level of transcription. According to the virus concerned, the early functions may be further divided into a few subclasses according to the times at which transcriptions are switchedon. The production of early messengerRNA (mRNA) is stopped or reduced some time after infection at the same time as late messengers are turned-on. These late messengersare responsible for the late functions. These comprise all those involved in building an infective virus particle, but often also those needed for the continued functioning of the cytoplasmic membrane of the infected cell. The production of mRNA, by all the viral genes involved in late functions, is switched on at the sametime; from now on all the late gene products are synthesised simultaneously, although at different rates. These genespecific rates are predetermined and, with very few exceptions, are not regulated by any type of feed-back mechanisms or interrelations between different gene products. For many viruses, in particular for DNA-containing bacteriophages,it has beenestablishedthat the building of a virion proceeds from a precursor particle known as the prehead (or previrion, provirion) to the final virion by passing through different steps of maturation. This morphogenesis follows, in general, a specific sequenceof events which used to be called the ‘morphogenetic pathway’. Many such pathways are enumeratedand discussedin an excellent review by S. Casjens and J. King [ 11. Such pathways should not be mistaken as representing a synchronous growth of all the particles built in one infected cell. As illustrated in figure 4, the assembly of a precursor particle is initiated randomly; each particle then individually follows the morphogenetic pathway. It is likely, but not proven, that the rate at which individual pathways are initiated dependson the concentration of a geneproduct still not identified. What is completely certain, however, is that the control of the pathway is not on the level of transcription. For a better understanding of this interesting situation let us now consider again the example of cells infected with bacteriophage T4. When we follow the synthesis of late gene products, we find that they proceed roughly linearly with time (figure 4). At 37’C and in a rich growth medium, enough proteins are made to produce about five phagesper minute. The time neededfor producing a phage particle is between 5 and 12 minutes; this maturation time can be measuredby feeding radioactively labelled amino acids to the infected cell, and following the appearance of radioactivity in mature phage particles. The result of such an experiment is shown in figure 5. These findings demonstrate that phage growth is by no means synchronous. Initiation of new particles proceedsrandomly in time and they mature independently of each other. In bacteriophage T4, for example, the type of ‘steady state growth’ described above can be made to last one to two hours by preventing infected cells from undergoing spontaneous lysis by use of mutants and suitable experimental procedures. 3

For several bacteriophages it is known that the morphogenesisproceeds by subassemblies.For T4 W. B. Wood and R. S. Edgar with their collaborators [21 have shown that tails, heads,and the two halvesof tail fibres each follow independent pathways (figure 6). The finished subassembliesjoin together to form mature virions. Tail morphogenesis proceeds from baseplateson which tubes and sheathsare assembledto give a complete tail; the head proceedsfrom a preheadwhich then maturesinto a finished head by several steps,which we will discussfurther below. One of these steps is the uptake of DNA into an intermediate precursor. This then matures sequentially, as shown by a progressive stabilisation. When finished, the head combines with a tail. In two separate pathways tail fibre-halves have been assembled,which then join to form complete fibres; six of these become attached to the headtail complex and so produce an infective virion.

of mutations affecting the major shell proteins, but of mutations of other form-determining (morphopoietic) genes. In figure 9 we show the morphogenetic pathway of the head of bacteriophage T4. The upper part of the figure shows a pathway that is consistent with all solid experimental data. The lower part shows a pathway which we have established experimentally for a given set of physiological conditions [61, but its generalvalidity has still to be proven. It was established with the aid of methods described in the following section. During the transformation of preheadsinto mature headsthe following events take place. First, one of the phage-codedproteins contained in the core of the preheadundergoesa cascadeof three consecutive stepsof partial proteolysis, leading to an enzyme called T4ppase [41. This enzyme acts in situ on nearly all the proteins of the prehead 131. It cleaves the

Figure 2 Scheme showing the reproductive cycle of virus infection exemplified by a bacterial virus. It shows precursors and intermediate particles, besides finished viruses. For more detailed discussion see figures 3 and 9. Viral proteins are green (tail) or blue (head). The viral DNA is red.

Some viruses show the interesting phenomenon of polymorphism: virus-related material can become assembledinto morphologically altered structures which are related to precursors, intermediate particles, or mature virions. Morphologic variants may occur spontaneously in small proportions among normal vii-ions, as a consequence of accidental error. Morphologic variants are also produced in large amounts as consequenceof mutations in some of the late genes,and even the host may play a role. Polymorphism is particularly accentuatedin bacteriophage T4: one example is the tail-sheath protein, which can become assembledin the form of polysheaths (figure 7). Another example is variation in the shape of the head, which is normally a slightly elongated derivative of an icosahedron. Two types of variants of this prolate head are of particular interest: the short (or isometric) and the giant head (figure 8). Thesevariants contain phage DNA which, upon infection, is injected into the bacterium. Becauseof its reduced size the isometric phage has an incomplete genome,while the giants contain severalcopies ofit. These variants occur as a consequenceof mutations in either the gene responsible for the major shell protein, or of other mutations or influences (see later). The available experimental evidencesuggests-but does not yet prove it for all cases-that this form variation is already determined at the level of the prehead. Other experimentally important form-variants of this bacteriophage are the polyheads, which are related to preheads (figure 10). Polyheads are abortive; that is, in vivo they cannot undergo further stages of maturation. Polyheads of phage T4 are not the result 4

major shell protein, the molecular weight of which is reduced from 59 to 47 kilodaltons. It cleaves also the proteins which compose the core. Second, the particle expandslinearly by about 15 per cent; this expansion is not accompanied by addition of subunits of the major shellprotein, and it is therefore conservative. Third, DNA is packagedinto the particle, most likely just before or during expansion, It is not yet established if it is obligatory for these three events to occur in this sequence;in particular, we do not know precisely into which precursor intermediate DNA becomespackaged or condensed.In this paper we will not be able to consider this very interesting step,which is a perfect example of a nucleic acid-protein interaction, but the interested reader can find a reviewing summary elsewhere[51. Methods for establishing morphogenesis

pathways

of particle

Assembly pathways are established according to the two following basic experimental methods: (a) A very elegant method was introduced by M. F. Jacobson and D. J. Baltimore 171and consists in following the flow of a radioactive label through particles which have been separated from each other by physical methods. Luckily, the different precursors and intermediate particles of viruses can, in many cases, be separated by sedimentation on sucrose gradients. When a radioactive pulse is given by feedinglabelled metabolitesto the infected cells, for l-2 minutes, the radioactivity of proteins appears first in the prehead; someminutes later this label appearsin

one or several speciesof intermediate particles [81. After further incubation this label becomes accumulated in the finished virion. Although very convincing at first sight, this method has, nevertheless,to be used with great care. The difficulties can easily be imagined when considering the steady-statetype of growth depicted in figure 4. If the pool of a given precursor-protein (A) is smaller than that of another (B), then the label of protein (A) may become integrated into a particle more efficiently than that of protein (B), the label of which becomesstrongly diluted in the pool. This effect might possibly simulate a wrong sequence in time. Another difficulty is associated with the necessityto open the cell in order to liberate and analyse the particles. Conditions of ions and small moleculeswithin the cell are in general different from those of the growth media and buffers used.

phage, partial penetration of the central tube of the tail, and injection of the DNA. 5 mn This micrograph (5 minutes after infection) shows the nuclear disruption induced by this phage. Normally this is accompanied by enzymatic hydrolysis of the host DNA. At this stage the DNA and its breakdown products are found mainly in the typical marginal vacuoles. At the same time the nucleotides of the host DNA are used for synthesising phage DNA, under the control of phage genes (early functions)At about 8 minutes after infection the structural proteins 15 mn used for assembling and maturing the phage start to be made. A few minutes later, the first infective phages appear. From now on phages become matured at a rate of about 5 per minute. This micrograph was taken 15 minutes after infection. It takes an average of 7 minutes to assemble and mature a particle. From results of indirect experiments it is generally believed that a prehead with no or only little DNA is made first (insert). This prehead matures into finished heads. In this process most of the prehead proteins are proteolytically processed (in situ partial proteolysis, leading in some of the proteins to a 5-20 per cent reduction of molecular weight) and the DNA becomes packaged. The maturing particle also undergoes an expansion, without needing a corresponding additional amount of protein subunits. The fine fibrillar material resembling the bacterial nucleus is now what iscalled ‘vegetative phage’; that is, replicating and transcribing phage DNA. This micrograph, taken after 30 minutes, shows simply a 30 mn later stage with an increased amount of finished heads, The amount of particles per section is about 1/20th of the amount of a complete cell.

Since precursors and intermediates have no compelling reason to be extremely stable particles, they might thus be liable to breakdown as a consequenceof cell lysis. Particles separated on sucrose gradients might be breakdown products instead of true native precursors or intermediates. 6OC 2 L5( 0' :

30( 15(

L Figure 3 Electron microscopy of the intracellular events accompanying the reproduction of bacteriophageT4. The infected cells are fixed by osmium tetroxide. They are postfixed in an aqueous solution of uranylacetate, which fixes DNA and stains nucleic acids and proteins. After dehydration the cells are embedded in a liquid epoxy resin. After curing, the solid blocks are sectioned to about 200-600 A, thickness. These are observed as such, or after poststaining with lead and/or uranyl sa1ts.fCourtes.y of Beate Menge, Jacomina v.d. Broek, H. Wunderli, K. Lickfeld, M. Wurtz, and E. Kellenberger.) 0 mn ThisE. colicell has still the morphology of an uninfected with the typical nucleus (less dark parts) of cells undergoing exponential growth. The infection starts after adsorption of the

cell.

Figure 4 Asynchrony of phage assembly. Schematic representation of events governing virus multiplication The upper curves represent the synthesis of early and late proteins and of viral DNA as it occurs in the case of bacteriophage T4. In the lower part the elongated, rectangular boxes represent the morphogenetic pathway undergone by individual particles: it is shown above enlarged, and with still more details in figure 9.

We have also to consider that the maturation time is relatively short. The number per cell of precursors or intermediate particles observed at any time, is therefore extremely small and estimated to be less than 10 particles per cell. It is thus quite obvious that pathways determined by this method have to be verified and completed by other observations, as we describenow. 5

(b) By using mutants in genesinvolved in maturation or by the action of drugs, the pathway might be stopped at determined steps.As a consequence,the particles prior to this step accumulate in the infected cell and appear in sufficient numbers for examination. By sucrose gradient centrifugation one can isolate and biochemically analyse them. By electron microscopy one can study their morphology. Any such particle, however, is not necessarily either a precursor or a breakdown product of a precursor. It could be an abortive product; in complex bacteriophages side-tracks of the pathway are frequently observed, and they result in particles which are unable to mature further. We have encounteredalready such an abortive particle, the polyhead, which occurs when one of the proteins involved in building a preheadis non-functional. For every observed particle one has thus to prove that it is maturable: by removing the block one should observe a decreasein their number with a concurrent increase of the particles which follow in the pathway. How is this crucial test of maturability performed experimentally? If a virus-related particle accumulates in the presence of a drug, then its eventual maturation is demonstrated if an equivalent amount of infective particles is produced after removal of the drug and in absenceof new protein synthesis(inhibited by chloramphenicol or puromicine).

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given by another host in which this suppressorgeneis not functional or absent. In the temperature sensitive (ts) mutants growth is as the wild type at 30°C (permissive conditions) but the mutation becomes ‘expressed’ when grown at 40.5 OC (non-permissive condition). Coldsensitive mutants can be isolated which grow permissively at 30°C and non-permissively at lower temperatures. HEAD 22.IPIII.20

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Figure 5 Time needed for building a bacteriophage T4. In this experiment radioactively labelled amino acids werefed for 2 minutes to a cell infected with bacteriophage T4. At variable times after this radioactive pulse, aliquot portions of the culture were taken, the cells opened with chloroform, and the released mature bacteriophages were harvested and purified. The radioactivity of these was determined and plotted as a function of the time of harvest after the pulse.

Figure 6 BacteriophageT4 assembly. This scheme (an updated version of that of Wood and Edgar [Z]) illustrates how this bacteriophage proceeds by joining subassemblies, which mature independently. The numbers besides the arrows denote the gene which is involved in the concerned step. gp23 is the product of gene 23, gp 23”the same protein after partial proteolysis (cleavage). This protein constitutes the shell of this virus. The cleavage occurs in situ, on the precursor particle, the prehead. The indication ‘host’ signifies that a structure of the host, e.g. the cytoplasmic membrane, is in some way involved.

Unfortunately, no drugs are as yet available which produce a complete stop at one single maturation step. In most cases the time lapse between one or a few steps is only prolonged. Even worse, abortive particles may be produced. Fortunately, we have another possibility open to us: bacteriophage is endowed with an easily accessible genetics, and ‘conditional lethal’ mutations are produced and can be isolated in nearly every gene 19, 11. Such mutations are indicated by the following property. Under permissive conditions particles are built which are infective and for all practical purposes, identical to the wild type. Under nonpermissive conditions the products of the mutated genes are altered, and they are non- or misfunctional. The permissive condition for so-called amber mutants (‘am’) is a given host which has a suppressor gene, while the non-permissive condition is

Amber mutations lead to a protein fragment of the gene product which-in nearly all known cases-is nonfunctional. It is a unique feature of amber mutants that the normal protein- as product of a given gene,is replacedby a fragment. By comparing the molecular weights one can thus identify the mutated gene which is coding for this protein. The introduction of the now widely used gel electrophoresis 1101opened completely new dimensionsto this research. Comparing the phage proteins produced by amber mutants grown under permissive and nonpermissiveconditions allows a geneticidentification 131. The ts mutations lead to an altered protein which, under non-permissive conditions, behaves like that of a lethal point mutation (change of an amino acid). They might be non-functional or misfunctional, and in contrast to ammutations which are nearly always non-functional, one has

mbn after

6

Figure 7 Bacteriophage T4 and polymorphism of its tail. BacteriophageT4 with its tail fibres, which promote adsorption, is shown in (a) and (b). In (a) we see also some phages with contracted tail sheaths, the same as are induced upon infection of a cell. Free tails and abnormal tail sheaths and tail tubes are also visible. (c) shows a tail, ready to join a head. (d) shows an isolated, contracted sheath and (e) a linear aggregation thereof. In (f). we see polysheaths which are assembled from the sheath protein-subunits (product of gene 18) when tail precursors, like baseplates with tailtubes are not present. The polysheath is a morphologic variant of the contracted sheath. (Micrographsfrom (a) the author: (b) to (e) Dr. M. Wurtz; (f) Dr. M. Yanagida.)

to study carefully each ts-mutant in respect of a possible misfunction. Sometimes proteins produced by ts-mutants at 40°C can become functional again, when the temperature is lowered to 30°C. At 40°C a precursor or intermediate particle can thus be accumulated which, after lowering the temperature, can then eventually become matured. In the case of mutational blocks of the maturation pathway one has obviously also to distinguish carefully between abortive and maturable particles. In the case of amber mutations, the maturability of accumulated particles is difficult to assess,but in the case of some ts mutants this is feasible by using the temperature-lowering experiments mentioned above. In all these experiments electron microscopy is obviously an essential tool for describing the morphology of the different particles considered. Besides this descriptive use of microscopy, the possibility of making particle counts, either in lysates or in sections, is most valuable 111, 121. In some cases, it is also possible to observe the content of an in situ

Figure 8 Morphologic variants of the head of bacteriophage T4. (a) shows three isometric variants besides a normal, prolate head; (b) and (c) show giant phages of different lengths. All these head variants contain DNA in amounts corresponding to the lengths of the head; they are all infective. (Electron micrographs (a) and (b) from Dr. F. Eiserling: (c) from 8. ten Heggeler.)

lysed cell spread on to a limited area of the specimensupporting film in the electron microscope. I this ‘in situ lysis’ particle counts per cell can be made and the maturability of a particle might become demonstrable 1131. Sequential events the bacteriophage

of the maturation head

of the shell

of

(a) Sequential conformational changes. In the pathways shown in figure 9 we have seen that bacteriophage T4 proceeds from the prehead (or r-particle) through an Eparticle into the final head. While the prehead is made of unprocessed proteins, those of both the shell and the core of the s-particle have now been processed by the T4ppase. By expansion, the shell of the e-particle then becomes transformed into the very stable capsid of the mature head. These sequential transformations of the shell offers the possibility of investigating the molecular mechanisms in some detail. It is well established that the shells of viruses are regular arrays of identical subunits; they are a kind of folded twodimensional crystals 1141. A. Klug and his group I 151 at Cambridge have developed special crystallographic methods which allow the study of such two-dimensional protein arrays by use of optical diffraction of electron 7

micrographs. Images can be reconstructed after the statistical ‘noise’ is filtered away. The image processingcan also be achieved digitally with a computer. These techniques are successful only when the number of unit cells is sufficiently large; a normally sized virus shell is too small for providing the required details. As we have seen General

pathway

prehead

assembly

of TL head head maturation

DNA-content demonstrated pathway

One experimentally core 21

action of this enzyme in vim; in vitro this proteolytic action can be achieved even on polyheads simply by adding the enzyme 1171.The second change is represented by the expansion. It is also still unknown by which mechanismit is triggered in vivo; in vitro it is induced by lowering the salt concentration of the buffer by a factor of lo- 100 1191.

T-particle prehead

stabilisation

-... +9---o-~

E-particle I-INA

core proteins *b

Pathway of bacteriophageT4-head maturation. Figure 9 The upper pathway accounts for all the experimental facts reported and which were obtained under various experimental conditions and/or different mutants. The lower pathway accounts for results under specific conditions and their general validity is not yet established. Completely finished cores without shells are observed in viva and in v;tro but demonstrated only in vitro to be maturable into preheads. The existence of the s-particle is demonstrated both in the case of mutants 16 and 17 and in presence of 9-amino-acridine. In the latter case, but not the former, its maturability in viva was shown. The conventions are the same as in figure 6: Numbers indicate those of the genes involved. IP’s are the geneproducts which lead after cleavage to three different internal proteins of this phage; the asterisk denotes that of these proteins 5-20% have been amputated by partial proteolysis (T4ppase).

above, bacteriophage T4 has two morphological variants that are elongated. The polyhead, which is a variant of the prehead,is suitable for in vitro processingwith T4ppase.Its shell undergoes in vitro the same maturation pathway as does that of a normal virion in vivo. The giant phages are variants of infective virions. The shellsof both thesevariant particles present a large enough surface area to allow a study of the so-called surface lattice by Klug’s crystallographic methods. It was, therefore, very challenging to follow in these variants the transformations undergone by the shell during the maturation of preheads into heads ] 16, 17 181. In figure 11 we summarise the results by showing the surface lattices of preheads (a); of the r-particle resulting after protein processing (b); and finally of the capsid resulting from expansion (c). Because of the still limited number of unit cells available and because of the deformations occurring during specimen preparation, the absolute information. obtained on the conformation is limited to some 30 A. The differences determined between the three lattices are, however, accurate to some 5 A. The experimentally determined changes of conformation are thus significant. The first change is due to the processingof proteins by the T4ppase. It is still unknown which mechanism triggers the sudden 8

(b) New binding sites are generatedfor interaction with othergeneproducts. The expanded, mature capsid (figures 1lc and 12A) is now able to bind other speciesof proteins picked out of the ‘soluble’ pool. Indeed, only the expandedlattice reacts with the minor proteins sot and hoc which T. Ishii and M. Yanagida [ 201had characterisedand localised on this page. As seenin figure 12, hoc binds in the middle of a capsomere and one sot binds to each protomer and is thus situated all around the capsomere121I. We have here a typical caseof a soluble protein not being activated by becoming modified, but being put into action (actuated) by changes occurring on the other reaction partner, the particle. Sot and hoc are present in the cell during the whole pathway of maturation of a particle; they do not interact with the major shell subunits either when these are still in the soluble protein pool (in the cellular sap or on the cytoplasmic membrane)or when they are already organised as precursor-shells. Interaction occurs only once the major shell subunits of the assembled particle have reached the proper conformation. By this conformational change sites are now generated for binding both sot and hoc. This reminds us very much of the so-called allosteric

enzymesdiscovered by Nobel Prize winner J. Monod. Here a conformational change is necessaryfor revealing the site necessaryfor binding of, and catalytically interacting with, a substrate. Contrary to the isosteric inhibition of an enzyme by binding an inhibitor instead of the substrate to the active site, the allosteric regulation involves another (allosteric) site, situated elsewhere on the protein. Interaction of this site with an effector induces the conformational changes which reveal the enzymatically active site. In many cases,the role of effector is played by the same protein species, when it polymerises into oligomers.

Figure 10 T4-prehead-polymorphism. (a) Shows normal preheads with their shell and inside core. (b) Shows polyheads besides normal phages. Preheads and polyheads are made of the still uncleaved gene product gp23. Preheads are further matured in viva into heads as described in figure 9 and in the text. Polyheads are abortive structures. ln vitro their shell can be made to undergo the lattice transformations correlated with the head maturation. (Micrographsfrom (a) Dr. R. van Driel, (b) the author.)

The minor shell proteins hoc and sot of phage T4 are dispensable; indeed, bacteriophage T2, which is its close relative, lacks them and mutants of T4 can be produced in which these proteins are also absent [201.In bacteriophage lambda, however, an indispensable shell protein, gpD, is involved in its maturation. On this phage the crystallographic studies cannot yet be made so precisely as in T4 because of some unfavourable symmetries of arrangement. The results 1221neverthelessillustrate very nicely the necessary conformational changes involved. In figure 13 we show the lattices of the prehead composed of gpE before and after expansion. Only after expansion can one molecule of gpD become bound to each molecule of gpE. This additionally bound protein is indispensable for stabilising the shell and thus for producing viable particles that are resistant to adverseenvironments.

These two examplesof bacteriophages,T4 and lambda, clearly demonstrate how gene products can suddenly become bound once a competent conformation of the proteins of the maturing particle is attained. At first no binding sites were available. Through maturation the proteins of the accepting particle have to undergo a conformational change in order to generatethe sites on the particle, which are then able to interact specifically with the additional, protein. (c) Discussion of the nature of these conformational changes governing the generation of speciJic sites of interaction. The data presented up to this point

demonstrates that the observed conformational changes certainly affect the quaternary structure. Changes of quaternary structure, such as those observed, might result solely from a simple rearrangement of protein subunits which rigidly maintain their tertiary structure. For the time being we have theoretical and experimental evidence, but no proof, that the tertiary structure of the subunits it also profoundly changed.The experimental evidenceis provided by the following two sets of experiments which involve labelling specific sites on the surface of the protein by Fab fragments of specific IgG antibodies and/or by observing the surface relief of both the inside and outside of the shell by using freeze-drying techniques instead of negative stain for preparing the specimen for electron microscopy 1231. The authors of these experimental data conclude that the observed changescan be explained only when at least one domain of the major shell protein moves in respect to others. In the broad sense,this is obviously a change of tertiary structure. The theoretical argument makes use of another important fact. It became obvious from studies on the partial or total dissociation of the involved particles that the sequential lattice changes are accompanied by a very strong increase of bonding between subunits. While the shells of preheads are in a still measurable equilibrium between ‘polymers’ and ‘monomers’, the latter are no longer detectable in the transformed lattice, where the bonding is so strong that it no longer makes much senseto speak of a chemical equilibrium. Since no covalent bonds are involved, one can explain such a strong hydrophobic interaction only by assumingthe simultaneousinvolvement of a large number of weak interactions. The strengthening of bonding is then explained by an enlarging of the areasof bonding or contact between subunits. It is most difficult to imagine such an increase of area without assuming rather substantial changes of conformation in the subunits themselves. Viruses provide particularly interesting examples of the final bonding between proteins being so strong that it has to be achieved in sequential steps. Assembly-or polymerisation-occurs with relatively weak bonding. Its increase is achieved in situ on the already fully organised shell by sequentialtransformations which, as we have seen, most probably involve profound changes of the tertiary structure. The shell of a virus is not the only example of such stabilisation occuring. In many bacteriophagesthe infective process is initiated by contraction of a tail-sheath, which promotes penetration of the tail tube through at least some of the layers of the cell envelopes; after that, DNA can becomeinjected. The contracted tail sheath’isnow the most strongly bonded and most stable structure of the phage.It is even more resistant to dissociation into subunits than the capsid! 9

This phenomenon of increased bonding is certainly worth further investigations. It seemsto us of the utmost importance to provide more data, with finer details of the structural changesinvolved.

becomeslarger the more subunits are involved in building a capsid. At first, this quasi-equivalence was thought to reflect only in the bonding angles between rigid subunits. Presently, we have learnedthat the interactions betweenthe

8 m aJ)N (a)

prehead

s

Fn

zl 2 ; z

E;:

(bl E - particle

Ek

;ii ii z U

(cl lw El 1 < Capsld ; (wlthout soc 8

hoc)

V

J.2

11.2nm h &9.6”‘0.7

nm I

qk3.5”+0.5

@=22”‘2.2

Figure 1 1 Lattice transformations correlated with maturation of the phage T4 head shell. These image-processed micrographs represent the lattices (a) of the prehead, (b) of the c-particle, (cl of the final stable capsid before sot and hoc are bound to it. The crystallographic analysis was done on particles as those represented in figure 8. Below the micrographs, a schematic drawing of the hexamerically arranged subunits is given. Between (a) and (b) the shell protein was proteolytically amputated byZO%and between (b) and(c) expansion occurred. (Courtesy Dr. A. Steven and Dr. J. Carracosa.)

Mechanisms of ‘form-determination’ ‘morphopoieseis’ (shape and size)

or

From theoretical arguments it has becomeobvious that the form of the shells of small icosahedral viruses might be the direct consequence of the shape and specific bonding properties of the constituting protein subunits [141.By self assemblyof the subunits the shapeand sizeof a shell would thus be unambiguously determined. This has been demonstrated to be true for the capsids of many small viruses. In such cases the information carried by the properties of the subunits is thus fully form determining. For larger shells it was argued that such a full form determining quality of the subunits becomes more and more problematic. Indeed a new principle, that of ‘quasiequivalence’,has had to be introduced 1141.It is necessarily involved in larger viruses, which have more than 60 identical subunits. Here the local environment of the subunits is not everywhere exactly the same when considering different locations on the surface lattice of the shell. The differently located subunits can no longer be related exactly to each other by symmetry operations. The necessary small deviations from precise symmetry leads to this quasi-equivalence. The required quasi-equivalence 10

subunits are in many casesvery strong and can be ruptured only under conditions which are such as to completely denature proteins. No covalent bonds being involved, we have thus to assume that bonding includes many amino acid residuesof the two partners, thus forming a relatively large area of such a hydrophobic interaction, as already discussed above. From chemical dissociation studies we have also learnedthat in most casesthe interactions are not detectably different between quasi-equivalently positioned subunits. We have thus to conclude that the sizes of the bonding areas are also not very different. Quasiequivalence is thus most likely reflected in conformational differencesin the subunits rather than in the bonding angles. From these considerations it was theoretically predicted that larger viruses and in particular all those which have no completeicosahedral symmetry as,for example,the prolate heads of bacteriophages T4 and @29 (reviewed in 33) would require additional form-determining mechanisms which complement the morphopoietic information carried by the major subunits of the shell. This was demonstrated by genetic data for phage T4 where numerous genes are involved in the form determination of a morphologically normal phage [24, 251. If one or a few of these gene

L = 13.0

L =13.0

L = 13.0

L = 13.0

D= 8.3

D=

D= 7.5

D=7.5

$=22"

#=

$=

21°

8.1

o=20°

22”

Figure 12 The addition of the minor proteinssoc and hoc to the T4-capsid. To the expanded, stabilised surface lattices in (A) and in figure 1 1 c. the minor proteins hoc and sot are now able to bind. (B) shows hoc bound in the centre of the capsomer. On (BC) and (C) increasing amounts of sot are bound. (These processed micrographs are due to efforts of Dr. U. Aebi, Dr. J. Kistler, (then graduate students) and Dr. A. Steven with the advice of Dr. M. Showe and Dr. R. P. Smith.)

products are not functional, form variants occur, as we have already. discussed above. We have also seen that bacteriophage preheads contain proteins inside the shell: they constitute the scaffolding, morphopoietic, or assembly core. According to the phage species in question, these cores are built by many copies of one or a few different sorts of proteins. For bacteriophages lambda and P22, it was demonstratedthat monsters are produced in vivo as soon as the core is absent or non-functional. These monsters have very variable forms and only a small proportion of the particles produced by a cell have the rounded isommetric form of the prehead.The core of bacteriophageT4 prehead contains at least five different proteins, of which two are dispensable. Absence of function of one or several of the essentialcore proteins leads hereto tubular forms. The results of in vivo experiments demonstrated that the form determination is shared by core and shell proteins. The relative amounts of information contributed by these two constituents towards form-determination of , the prehead is different for different species. In the case of phage T4 the core carries a particularly large amount of form-determining information, as has been demonstrated by the in vitro experimentsby R. van Driel et al. 126,27 281. Core and shell proteins have been separated by various biochemical procedures. Shell protein alone can become reassembledinto tubes. Core proteins alone give naked, prolate cores. A mixture of both core and shell proteins leads to prolate preheads.Naked cores can be completedto form preheads by addition of the shell protein. Hence we conclude that the shell protein has the capacity only for determining the lattice and its curvature, thus leading to tubes. The information for rounding-up and building a closedprolate shell is provided by the core. We have mentioned above that phage T4 is amenableto producing form-variants and among them, the short isommetric head and the very elongated giant one. All hitherto available experimental evidence suggeststhat this form determination had already occurred at the level of the prehead. It is, however, not yet fully excluded that these variants might follow alternative pathways, different from those established for normal phage and discussed above (figure 9).

L-

lO.Bnm

L = 13.0nm

L=

13.0nm

Lattice transformations during maturation of the shell Figure 13 of bacteriophage These processed micrographs show the negatively stained lattices of the prehead (A), constituted only of gpE; in (B) it is expanded and has generated binding sites for gpD. This latter binds in equimolar amounts to gpE as shown on (C) and figure 14. The crystallographic tubular variants of the analysis was done on in vitro reassembled prehead. The bright spots on (C) are trimers of gpD, which protrude from the surface as illustrated in figure 14. (Courtesy Dr. M. Wurtz and Drs. Th. and B. Hahn.)

Definitive and consistent data are not yet available for understanding the mechanismsgoverning the occurrence of these form variants. Some working hypothesis or models have been published. In agreement also with other observations 1291,M. K. Showe and L. Onorato 1301have formulated the kinetic model of form determination: during assembly of a prehead its core and shell would grow simultaneously. According to the ratios of their rates of growth, short, normal, or long heads would be produced. Since these rates of growth are expected to depend on the concentrations of the corresponding soluble subunits in the pool, this model can now be checked experimentally in vivo and in vitro by adequately varying the pool sizes. Not all observations are as yet consistent with this elegant kinetic model: in our laboratory Franz Traub has found intracellular particles which seemto be ‘naked’ cores when the shell protein is not madeavailable. This suggestsa possible precursor role for cores. These might become assembledalready possessingthe correct form, before the shell is built around them. This observation is in agreement with the in vitro experiments described above in which the core can become surrounded by the shell in a later step. Both results suggest that the form and particularly the length of heads are already determined by their cores. Further experiments are neededto resolve these apparent contradictions. Even if the kinetic model is not verified for the role of core and shell, it would still keep its general interest as a basic mechanism.Indeed, the size and length of the core could be determined by the ratios of the growth rates of two core constituents instead of by those of core and shell. Conclusions

and Outlook.

From what we have described above it is clear that the study of phage morphogenesis has provided proofs for a 11

control mechanism governing sequential protein-protein interactions. Conformational changes in the maturing particle are required in order to promote its interaction with a protein which was ‘waiting’ in a pool. This mechanism is comparable to the allosteric regulation by which a conformational change in an enzyme is required for interacting with, or acting on, a substrate. The maturing particle would correspond to the enzyme, the additional, new subunits to the substrate. By analogy to this mechanism of enzyme activity, we proposed a mechanism of control of protein-protein interactions by sequentially induced conformational changes earlier (see 1311.for the first explicit formulation) and it is rewarding to seethat it has been experimentally verified. For the student of phage morphogenesisit is obvious that many more examplesare available in which this sort of control is likely to be involved. We would like to mention particularly the phage tail, the morphogenesis of which has been extensively studied by the group around King [I I.

phage head is in large measuredetermined by a scaffolding core. The form determining contribution of the shell protein comprises in all cases a capacity to form a lattice. In addition, the subunits have an intrinsic property“which leads to a curvature of this lattice. According to the species of virus considered, this information may either be strong enough to produce a closed shell or be sufficient only to make cylinders. The hitherto accumulated data show that the methodology applied for the study of phage head morphogenesisis potentially able to answer positively the remaining questions on form determination, particularly those of length, in the not too far future. Another very challenging object of ongoing research is the length determination of the phage tail. Despite numerous attempts, it has not yet been possible to decide between various proposed mechanisms of length determination 1321. Among those, only two remain as testablealternatives. In one, a fibrous protein of determined length is usedas a sort of measuringdevice.In the second,a

Figure 14 Artists represenfation of the lattice transformation of the capsid of bacteriophage This picture illustrates the situation shown in figure 13 and shows the addition of gpD to the expanded lattice of the shell made of gpE. (Courtesy Dr. M. Wurtz.)

The methodology which has beendevelopedfor studying two-dimensional protein-surface lattices and their conformational changes is immediately applicable to ordered lipoprotein membranes. We have shown, on the example of the maturation of a virus shell, that conformational differences down to some 5 A can be determinedin relative terms by using conventional methods of electron microscopy. Thus it should also become possible to detect conformational changes of ordered membrane proteins. The biological interest of this methodology is obvious when we think on the still unknown mechanisms controlling opening and closing of gates or pores, active transport, and signal transmission in biological membranes. The problem of form determination, although of more academic interest, was always a challenge to the human mind. We believe that here also phage has provided some clues. We have seenthat the determination of the length of a 12

cooperative phenomenon of ‘cumulated strain’ would lead to a termination of a quaternary structure after a given number of subunits has become assembled1311.In such a model, one has to assume that the conformation of all subunits suddenly changes in such a fashion that no interaction sites remain available for further elongation of the structure. All in all, the results set out in this paper can be comfortably explained by assuming relatively flexible and malleable proteins which can easily be induced to change their tertiary structure. They would thus representthe other extreme to the relatively rigid proteins represented by enzymes, the activity of which is dependent on only very small conformational changes. Structural phage proteins would share this property of high malleability and susceptibility to large conformational changeswith someof the membraneproteins. The classification of proteins betweentwo extremeswith

rigid and malleable properties is an interesting and provocative thought. Could it be that the rigid proteins, by their nature, could easily be crystallised into threedimensional crystals accessible to X-ray diffraction analysis, while the more flexible proteins, again by their nature, would be more resistant to crystallisation? If this were shown to be true, then the study of two-dimensional arrays by means of crystallographic techniques applied to electron micrographs would be a very important addition to the already available methods. We hope that these considerations will encourage investigators to consider bacteriophage as a useful model for the study of such, still hypothetical, malleable proteins and of control mechanisms based on conformational changes. These possibilities are, in my opinion, entirely sufficient to justify the study of bacteriophage and to encourage young people to enter this field, despite the fact that they have to cope with an enormous wealth of experimental observations already available, and in spite of the uselessnessof phagein medicine and society!

References

111 Casj;;;7fj

and King, J. Ann. Rev. of Biochemistry

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121 Wood, W. B. Edgar, R. S. King, J. Lielausis and I. Henninger, M. Fed. Proceedings 21.1160. 1968. 131 Laemmli,U. K.N&reLond. 227,680,1970. 141 Showe. M. K. Isobe, E.. and Onorato. L.J. Mol. Biol. 107.35, 1976. [5] Kellenberger, E. BtoSystems: Journal of Molecular, Cellular and Behavioral Origins and Evolution, in press. 161 Schiirli, E. and Kellenberger, E. J. Virol, in press. 171Jacobson, M. F. and Baltimore, D. J. Mol. Biol. 33,369,1968. i81 Laemmli, U. K. and Favre, M. J. Mol. Biol. 80,575,1973. 191 Epstein, R. H., Bolle, A., Steinberg, C. M. Kellenberger, E., Boy de la Tour, E., Chevallier, R., Edgar, R. S., Susman,M., Denhardt,

Endmvour, NwvS~rio~Volurm4, (0 Pebqamon Pmu. Ptintodin

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1101 Shapiro, A. L., Vinuela, E., and Maizel, J. V. Biophys. Biochem. Res. Comm. 28,8 15,1967.

[ 111 Lickfeld, K. G., Menge, B., Wunderli, H., van den Broek, J., and Kellenberger, E. J. UltrastructureResearch 60, 148, 1977. [ 121 Kellenberger, E. and Bitter%,D. Microscopica Acta 78,13 1, 1976. [131 Bijlenga, R. K. L., van den Broek, R., and Kellenberger, E. J. Supramol. Struct. 2,45,1974.

I141 Caspar, D. L. D. and Klug, A. Cold Spring Harbor Symp. on Quant. Biol. 21,1, 1962. 1151 Crowther, R. A. and Klug, A. Ann. Rev. Biochem. 44,16 1,1975. 1161 Laemmli,U.K.,Amos,L.A.,andKlug,A.Cell7,191,1976. 1171 Steven, A. C., Couture, E., Aebi, U. and Showe, M. K. J. Mol. Biol. 106,187,1976. 1181 Steven,A. Carrascosa, J. J. Supramol. Struct. 10,1,1979. I191 Carrascosa. J. L. J. Virol26.420. 1978. 1201 Ishii,T. and’yanagida, M. J.Mol:Biol. 97,655, 1975. 1211 Aebi, U, van Driel, R., Bijlenga, R. K. L., ten Heggeler, B., van den Broek, R., Steven, A.. and Smith. P. R. J. Mol. Biol. 110.687. 1977. 1221 Wurtz, M., Kistler, J., and Hohn, T. J. Mol. Biol. 101,39, 1976. 1231 Kistler, J., Aebi, U., Onorato, L., ten Heggeler, B., and Showe, M. R. J.Mol. Biol. 126,571, 1978. 1241 Kellenberger, E. ‘Principles of Biomolecular Organisations’ (G. E. W. Wolstenholm and M. O’Connor, (eds)) pp 192-226 Churchill, London. 1966. 1251 Kellenberger, E. in: ‘Symmetry and function of biological systems at the macromolecular level’. (A. Engstrom and B. Strandberg (eds) ) Pp 349-366. Almqist and Wik&ll, Stockholm, 1969. [261 van Driel, R. J.Mol. Biol. 114.61. 1977. 1271 van Driel, R. and Couture, E.J. Mol. Biol. 123,7 13, 1978. 1281 van Driel, R. and Couture, E. J. Mol. Biol. 123, 115, 1978. 1291 Yanagida, M., ‘Proceedings of 1977 Molecular Biology Meeting, Japan’, Kyoritsu, Shuppan Co., Tokyo 1978. 1301 Showe, M. K. and Onorato, L. Proc. Natl. Acad. Sci. USA 75, 4165,1978. 1311 Kellenberger, E. in: ‘Polymerisation reactions in biological systems’ (G. E. W. Wolstenholm (ed) ) pp 189-206, 295-298. Ass. Scientific Publ., Amsterdam. 1972. 1321 Kellenberger. - E. Phil. Trans. R. Sot. Lond. B. 216.27. 1976. 1331 Butler, P. J. G. Int.Rev. Biochem. 25,205,1979.

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